Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which contributes to the pathogenesis of human cancers. Hence, it is of paramount importance to discover and interrogate novel key constituents of the mammalian DNA damage response network. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the essence of an intact S-phase checkpoint in maintaining genome integrity. Although basic framework of the S-phase checkpoint in multi-cellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome band 11q23 translocation disrupting the MLL gene results in poor prognostic leukemias that carry pathognomonic MLL fusions. MLL is a transcription co-activator that is best known to maintain HOX gene expression. The importance of HOXA gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models indicated that MLL fusion-induced HOXA gene aberration alone is insufficient to initiate MLL leukemia. Therefore, further dysregulation must exit and contribute to the ultimate leukemia phenotype. Our recent studies demonstrated a close relationship between MLL and the regulation of mammalian cell cycle. MLL not only assists in the G1/S and G2/M phase transition during a normal cell division cycle but also executes the S-phase checkpoint upon DNA damage. We found that (1) MLL functions as a key effector of ATR-mediated S-phase checkpoint response, (2) activated ATR phosphorylates and thus stabilizes MLL, (3) upon checkpoint activation MLL accumulates at the late replication origin, methylates histone H3K4, and thus delays DNA replication, (4) MLL deficient cells exhibit defects in the S-phase checkpoint response, and (5) MLL fusions work as dominant negative mutants that compromise the integrity of S-phase checkpoint. Here we will determine the mechanisms by which MLL executes the S phase checkpoint response and examine whether and to what extent an S-phase checkpoint dysfunction contributes to MLL leukemogenesis. Our proposal connects MLL/MLL fusions to the S-phase checkpoint response network, which not only provides novel insights into the mammalian cell cycle checkpoint control but also shed light on the pathogenesis of poor prognostic human leukemias. PUBLIC HEALTH RELEVANCE: Cancer is a public health issue that directly impacts millions of lives and costs billions of dollars in the United States each year. Through a better understanding of the molecular pathogenesis of cancer, targeted therapeutics may be developed and eventually benefit advanced- stage cancer patients (1-2). Mixed lineage leukemia, resulted from chromosome translocation of the MLL gene, portends poor prognosis (3-6). Consequently, novel treatment strategies for this dreadful illness are urgently needed. My laboratory over the years has helped elucidate novel regulations and functions of the MLL protein (7-14). Cell cycle checkpoints safeguard our genome and compromised checkpoints contribute to the evolution of human cancer (15-18). Hence, we are particularly excited about our recent studies that link MLL to the DNA damage response network (14). Based on these findings, we propose to further investigate how MLL regulates the S-phase checkpoint and determine whether checkpoint dysfunction contributes to the MLL leukemogenesis. Data obtained from this current grant expect to provide novel insights concerning the mammalian DNA damage response network and shed light on the molecular pathogenesis of MLL leukemias.